Production of alpha-1,6-glucosidases

ABSTRACT

The production of alpha-1,6-glucosidase by the cultivation of a strain of Klebsiella pneumoniae ATCC 13883, Klebsiella rhinoscleromatis ATCC 13884, Klebsiella ozaenae ATCC 13885, Klebsiella edwardsii ATCC 13886 or Klebsiella rubiacearum ATCC 15574 on media consisting of carbon sources, nitrogen sources, inorganic salts and others under aerating and agitating conditions thereby resulting in accumulation of alpha-1,6glucosidase on the media and the separation of the alpha-1,6glucosidase from the media following the purification of the alpha-1,6-glucosidase.

United States Patent n91 Masuda et al.

[ PRODUCTION OF ALPHA- l ,G-GLUCOSIDASES [75] Inventors: Kazuo Masuda; Kaname Sugimoto,

both of Okayama, Japan [73] Assignee: Hayashibara Company,

Okayama-shi, Okayama, Japan [22] Filed: Apr. 23, 1971 [21] Appl. No.: 137,023

[30] Foreign Application Priority Data Apr. 25, 1970 Japan 45/35712 [52] US. Cl. 195/66 R, 195/65 [51] lint. C1 C07g 7/028 [58] Field of Search 195/62, 66

[56] References Cited UNITED STATES PATENTS 3,632,475 1/1972 Sugimoto et a1 195/31 R OTHER PUBLICATIONS Breed, et al., Betgeys Manual of Determinative Bacte- 11] 3,766,014 Oct. 16, 1973 riology, 7th Ed. The Williams & Wilkins Co., 1957 (PP- 344-346) QR8156C.2.

Veda, et al., Production of lsoamylase by Escherichia Intermedia, Applied Microbiology, May 1967, Vol. 15, No. 3 (PP. 492-496) QR1A6.

Primary Examiner-David M. Naff Att0rneyBrowdy & Neimark [5 7] ABSTRACT The production of alpha-1,6-glucosidase by the culti vation of a strain of Klebsiella pneumoniae ATCC 13883, Klebsiella rhinoscleromatis ATCC 13884, Klebsiella ozaenae ATCC 13885, Kle'bsiella edwardsii ATCC 13886 or Klebsiella rubiacearum ATCC 15574 on media consisting of carbon sources, nitrogen sources, inorganic salts and others under aerating and agitating conditions thereby resulting in accumulation of alpha-1,6-g1ucosidase on the media and the separation of the alpha-1,6-g1ucosidase from the media following the purification of the alpha-1,6-glucosidase.

2 Claims, No Drawings The present invention relates to a process for the production of alpha-1,6-glucosidases, comprising cultivadine reaction with the addition of a N/ 100 l -Kl solution were recorded.

To another test tube of those cultures was added 0.2 percent SDS solution (containing 2 ml Mc Ilvaine were diluted 10 times and then the optical densities were read using a spectrophotometer (Hitachi Model 101) and a photocell 1 cm wide at 660 mu. Further, the iodine colors of the test solutions obtained by the iotion of Strains of the genus Klebsiellfl- 5 buffer, pH 7.0, per 5 ml 0.2 percent SDS solution) Enzymes that hydrolyze specifically the alpha-l, autolyzed at 28C for 30 hours in a test tube shaker, glucosldlc bonds of Starch, g y g were defined and then the enzymatic activity was determined. as lsoamylases- However, Slhce other enzymes with Enzyme activity was determined by the following similar a t O a d c pa l of hy r yzmg the ph procedure. A reaction solution comprising 5 ml of l 1,6-glucosidic bonds of pullulan were found later in 10 .percent soluble glutinous rice starch, 1 ml of N/2 ace- Aerobacter aerogenes the enzymes were designated pultat buffer H 6.0), and 1 ml of said SDS autolyzate lulanase and have been utilized in studies on polysacwas incubated at 40C. Prior to and 30 minutes after charides. With the purpose of utilizing the hydrolyza be g inn ing the incubation0.5 ml fragtionsof the reac-i tion action of the enzymes in commercial production of tion solrition were collected and poured into "l5ml of starch hydrolyzates, the inventors screened alpha-1,6- 'N/50 H 80 After discontinuation of the reaction to glucosidase producing strains selected from a wide vathe resulting solutions were added 0.5 ml of N/ 100 riety of type cultures and newly isolated from soils rel -Kl and developed. 15 minutes later the fractions sulting in the discovery of superior strains from genera were determined using Hitachi Model 101 spectrophoof Esherichia, Pseudomonas, Actinomycetes, Lactobaciltometer and a cell 1 cm wide at 610 mu. An optical lus and others. By the evaluation of type cultures of the density increase of 0.100 measured at the beginning of genus Klebsiella, the inventors obtained high activity the reaction and minutes later was designated 10 enzymes from five strains. fu/ml.

TABLE 1 Iodine reaction pH O.D. at 660 m FuJml.

3 5 10 3 5 10 a 5 10 Number Strains 3 days 5 days 10 days days days days days days days days days days 1 KlgbgsgigllapneumoniaaATCC Aohromic. Achromic. Achromic. 8.0 7.9 7.8 0.475 0.414 0.318 18.6 27.6 21.4

1 2 Klebsiella rhinoscleromatis, Blue Bluish Violet 8.6 8.4 8.5 0.423 0.398 0.277 21.0 22.0 6.6

ATCC 13884. violet. 3 Kiglgsiella ozaenae, ATCC .do ..do. Regglesgt 8.6 8.3 8.2 0.364 0.389 0.397 15.2 19.8 8.0 4 Kiealgxsiella edwtzrdsii, ATCC .d0 do Achromic. 8.7 8.4 7.8 0.308 0.320 0.348 17.2 18.4 9.8

6. 5 Klehsiella rubiacearum, Bluish Reddish do 8.4 5.4 7.1 0.227 1.076 0.571 23.0 41.6 25.4

ATCC 15574. violet. violet.

' The strains in which the inventors found enzymatic To the said culture broth incubated for four days was activity were added SDS (Sodium Lauryl Sulfate) and autolyzed for y a day. Fractions of (NH.,) 80 0.2 0.7 saturation, Klebsiella pneumoniae ATCC 13883 mebsieua rhinosdemmafis ATCC 13884 were collected, dialyzed against tap water, 0.02 N ace Klebsiella ozaenae ATCC 33 tate buffer was added, and then their enzymatlc activity Klebsiella edwfl'dsii ATCC 13886 and action on amylose or soluble glutinous rice starch Klebriella rubiacearum ATCC 15574 were investigated. In the specific case of Klebsiella ozaenae, the enzyme A common culture medium comprised peptone and acts and hydrolyzes nearly 30 percent of the amylose, yeast extracts, and to whlch were added liquefied which suggests a possibility of co-exlstence of alphastarch as a carbon source, potassium phosphate, dibaa amylase in the enzyme solution. Thus the color develsic magnesium sulfate and ferrous sulfate as inorganic opment by Iodine is extremely slight, showing a CD. of salts. On the medium were inoculated strains grown on o 0.016. No co-existence of other types of amylases were a bouillon agar slant, then cultivated at 28 C in test noticed in the rest of the enzymes. The enzymatic actubes under shaking conditions for 10 days. A summary trons increased iodine strains with the sole exception of of the results is given in Table 1. Each strains exhibited Klebszella ozaenae (listed No. 2 m Table l), exhlbitmg enzymatic activities of 15 25 units, thus by selecting their h dml zin ca acmes on M ha 1 6 lucosidic desirable media, an acquisition of enzymes with considbonds y y g p p g erable activities may be expected.

, Upon mvestlgatmg the properties of such enzymes A A IS r the optimal pH for an enzymatic reaction at 40 C using N LYS soluble glutinous rice starch as a substrate was found to Two cultured test tubes of each strain were collected be pH 5.5 6.0 by determining the increase in optical 3, 5, 7 and 10 days after beginning the cultivation. One density of iodine-color. Studies on the increase of optitube of those cultures was autoclaved at 120C for 20 cal density of iodine color obtained from 20 hours reacminutes and its pH, optical density and iodine color (iotion at pH 6.0 and at various temperatures showed that dine reaction) were determined. the optimal temperature for the enzymatic reaction was Determination of pH: analyzed with the employment 45- 50C. However in the tests for Klebsiella azaenae of a pH meter (Hitachi-l-loriba M-5 Model) the incubation was conducted at 55C in order to inac- Determination of optical density: the culture broths .tivate the other types of amylase activity by heating.

Tests on thermal stabilities treating the enzymes for one hour in the absence of substrates showed that the enzymes were stable at 40C, while most of the activities were inactivated at 50C.

The substrate specificities were investigated resulting Liquefied starch l in the findings that each enzyme hydrolyzed 100 perzig z i 6? cent of waxy corn starch in the co-existence of betaamylases and the hydrolysis rate of the enzymes on poo 36 tato starch, waxy corn starch and waxy corn starch 5 so m b Ioo beta-limit dextrin were 90 100 percent. Each enzyme reacted on pullulan causing hydrolysis of 75 95 Culture;

percent, displaying specificities identical to those of pullulanases. Strains of Klebsiella ozaenae were the sole enzymes which possess no hydrolysis action on glyco- 10 gen, thus the strains were conceived to be an enzyme with different mechanism of action.

v The enzymes derived from the above described organisms generally possess properties similar to those of pullulanases and possess considerably high activities and in addition are capable of hydrolyzing pullulan and glycogen, as well as starches. However Klebsiella ozaenae is the sole exception and is incapable of hydrolysis of glycogen.

The present invention will be described with illustrative of examples. The percentages and parts used hereinafter are by weight unless stated otherwise. It should be understood that no undue limitations or restrictions are to be imposed by the examples, but that the present invention is defined by the appended claims.

EXAMPLE 0 (0- 2C, 8,000 r.p.m., 10,000 X G, for 20 minutes).

The collected precipitates were dialyzed against tap warei s c, for two d a ys an cehtrifu'ged fiml 13,500 r.p.m., 23,000 X G, for 10 minutes). Furthermore the solution removed of precipitates from the dialyzed enzyme solution was fractionated by adding (NHQ SO to 0.2 0.7 saturation. The precipitates of this fraction obtained by the centrifuge were dialyzed against tap water, 8C, for 2 days, and acetate buffer was added with a final concentration of 0.02 N, and

thus the solution was used as the test enzyme solution.

The results of testing these enzyme solutions for enzymatic activities and their action on soluble glutinous rice starch* or amylose** are given in the following table.

Action on amylose (incubation at 40 C.

for 20 hrs.)

Action on soluble glutinous rice starch Activity Decomof enzyme position Blue Optical density and increment in optical density solution rate, value at Number Strains used percent 680 ma 0 hr. 0.5 hr. 20 hr.

1 K lcbsiella pneumoniae, ATCC 13883 22. 5 2.1 0. 639 0.361 (0) 0. 586 (0. 225) 0.925 (0. 664) 2..- Klcbsiella rhinoscleromatis, ATCC 13884". 17. 1.5 0.318 0.371 0.540 50.169) 0.960 (0. 589) 3 K lebsiella ozaenac, ATCC 13885 15. 27.2 0.016 0. 346 0. 480 0.134) 0.080 (-0, 266) 4 Klebsz'clla edwardsii, ATCC 13886. 17. 1.7 0.290 0.431 0.500 (0. 078) 0.920 (0.480) 5 Klebsiella rubiacearum, ATCC 15574 11. 1. 6 0. 241 0. 364 (0) 0. 474 (0. 110) 0.740 (0. 376) Control Aerobact r eroyen AT 96215 NOTE: I

1 Reaction 0 hr., 1.310. 1

6 Enzyme solutions fractionated by saturation to 0.2-0.7 with (NH4)2S04 were used. 0 In the table the error of each hydrolysis rate is expressed percent values (given in the table) *0.20.5%.

'essrysecmmieconesaonybn mam bananas agar media were employed in the examples.

Medium; W 7

100 ml of the following medium composition was poured into 500 ml Erlenmeyer flasks sterilized according to the usual procedures by dry heating and auto- 65 claved at 120C for 20 minutes and used as medium.

" The action on soluble glutinous rice starch was determined by the following procedure. A mixture comprising 0.5 ml 1 percent soluble glutinous rice starch, 0.1 ml 1 percent N/2 acetate buffer (pH 6.0). and 0.1 ml of said enzyme solution was reacted at 40C for 20 hours. and fractions of 0.1 ml each were collected at 0, 0.5 and 20 hours. The incubation was discontinued with an addition of 3 ml of N/SO H.50 and developed with 0.1 ml of N/100 1,-Kl solution. 15 minutes later thegptical densities were determined at 610 mg. with 1 cm cells.

The optical densities of color developed solutions obtained by diluting the 20 hours reaction solutions were measured comparing a color developing agent (comprising 0.10 ml 20 hours reaction solution, 17.07 mi N/50 11,80 and 0.72 ml 0.2 1,-2 percent Kl solution) with those of control (comprising 0.10 ml pure water, 17.07 mi N/SO 11,80 and 0.72

ml 0.2 1,-2 percent Kl solution) prepared individually for each test solution immediately before the determination) using 1 cm cells and a 'spectrophotometer. Hitachi Model 124 at 380-800 ma. The color i developing agent employed was the one according to E. .l. Boume, W. N. Haworth, A. Macey and S. Peat, (.1. Chem. Soc. 1948, 924) and with a concentration of four times.

" The action on amylose was determined with the employment of 0.4 percent amylose (an amylose solution dissolved with N-NaOl-l, and neutralized with N-HCl, a product manufactured and distributed by Nagase Sangyo lfiabushiki ltaisha). A mixture comprising 2 ml of 0.4 percent of the test solutions (each contains 0.02 N acetate buffer, pH 6.0) was incubated at 40C for 20 hours. The amounts of direct reducing sugars and total sugars were analyzed quantitatively by the Somogyi-Nelson method and the Anthrone method. The increase obtained 20 hours after the commencement of the reaction was expressed by dextrin was incubated for three days.) Enzyme activity of said betaamylase was determined by the following procedure.

A reaction solution comprising 5 mi of l ercent soluble starch, 4 ml of M/lO acetate buffer (pH 5.0), 1 ml enzyme solution was incubated at 40C. After discontinuation of the reaction, direct reducing sugars produced under the above conditions were quantitatively anamulliplying 100 to the hydrolysis fate calculated direct reducing sugars ,lyzed by Fehling-Lehmann-Schoori's method. An increase of i0 mg of g tolal g l irect reducing sugars (as maltose) after incubation at 40C for 30 min- 2. The Properties of the Enzyme uteri was gn ed 1 u/ml.

In the case pulluian was used as substrate beta-amylase was replaced Usmg Said soluuions the effects of PH f ff with pure water. l0 u/ml of betaamylase solution was used in the case tures on the reaction rate and thermal stabrhtres were of Klebriella ozaenae ATCC 13885. determined. The optimal pH, optimal temperatures and Mlllhifit? O 19 383 q s e Q1 ai lmlfi i li lfl solution was used. sifabllmes w mvesngated' The results of the in only the case of Klebsiella ozaenae ATCC 13885 the incubation investigation are listed in the following table. was effected at 55C.

Optimal Thermal stability*** tempera- (residual activity) percent Optimal ture, N0. Strains pH 0. 4 C. 30 C. 40 C. 50 C. 1 Klebsz'ella pneumom'ue, ATCC 13883 5.5 50 100 100 90 0 2. Klebsiella rhinosclercmatis, ATCC 13884.. 6.0 50 100 100 92 0 3. Klebsiella ozaenae, ATCC 13885 6.0 50 100 100 100 0 4.. Klebsiella edwardsii, ATCC 13886.. 6.0 45 100 100 94 0 5 Klebsiella rubiacearum, ATCC 15574. 6.0 45 100 100 100 0 Control Aerobacter aerogenes, ATCC 9621 6. 0 50 100 100 95 2 NOTE Direct reducing sugars were determined by Somogyi-Nelsons The optimal pH was determined according to the above described method, while total sugars by the Anthrone method. The rates of hymethod for the determination of iodine activities using M/2 acetate drolysis were expressed in the case pullulan was used as substrate as buffers of pH 5.5 and 6.0 and M/S phosphate buffers of pH 6.0, 6.5 and maltotrroseltot ga X 100 (P whereas for the other 8017' 7.0 as buffers and said enzyme solutions, reacting the mixture at smile as alt /to S g X 100 (P 40C**and determining the increases of optical densities at 0.5 and V. 20 hours to those at 0 hour. The results are given in the following table.

Enzyme fl-amylasc plus K lebsiella fl-arnylase plus [EX-amylase plus fi-arnylass plus Klcbsiella rhinoscleklebsiella Klabsiella Klebsiella pneumoniae, romatis, ozaenae, edwardsii, rubzacearum, Number Substrate B-amylase ATCC 13883 ATCC 13884 ATCC 13885 ATCC 13886 ATCC 15574 1 Potato starch, percent 64 96 96 107 89 8 2.. Waxy corn starch, percent..." 67 101 101 106 101 9 3.. Waxy corn starch B-limit dextrin, percent 0 89 94 93 89 81 4" Oyster glycogen, percent 41 72 75 49 67 98 6 Pullulan, percent 0 74 92 82 94 77 Tlie optimaitemperatures were also determined according to the ffi fiar t tablafe ivfi r e sfilfi gbt a ined by method for iodine activities, reacting the mixtures at pH 6.0 and at b O 5 l 1 b temperatures of C, C, 50C, 55 c and 60C and determining the atmg a mixture compflslng m Percent increases of optical densities at 0.5 and 20 hours to those at 0 hour. strate solution used in the above investigation on subrreisaraarsasiisitzwere?aaersrirawtrarasamgarises 40 strate specificity, 0.1 ml N/2 acetate buffer (pH 6.0)

dure. After treating said enzymesolutions, to which 0.02 N acetate buffer was added, for one hour at 4C, 30C, 40C, C, C, C

and C, the residual activities were determined, according to the ioand 0.1 ml enzyme solution (4 fu/ml) at 40C for 20 hours.

Enzyme Klebsiella Klebsiella rhinoscleroma- Klebsiella Klebsiella Klebsiella pneumoniae, tis, ATCC ozaenae, edwardsii, rubiacsrum, ATCC 13883 13884 ATCC 13885 ATCC 13886 ATCC 15574 Number Substrate Hydrolysis Rate (as glucose) percent 1 Potato starch 0 2 12 3 11 2.- Waxy corn starch 2 2 14 4 7 3-. Waxy corn starch limit dextrin- 7 9 21 11 14 4.. Oyster glycogen l l 10 2 2 5 Pullulan 26 32 29 33 27 d i activfi measurement method. NOTE A beta-amylase extracted from wheat bran, treated with an acid, fractionated with ammonium sulfate and dialyzed, was used in the investi gation. (No variation was noticed on the iodine color of reaction mixture when mixture of said beta-amylase solution and potato beta-limit Each substrate was incubated solely with the above enzyme solutions from the strains of the genus lfilebsiella.

W Direct reducing sugars were quantitatively determined by Somogyi- Nelson's method while total sugars by the .Anthrone method. The hydrolysis rates were expressed as total reducing sugars (as glucose)/total sugars X I00 (percent).

The error of each hydrolysis rate is in value (give in the table) i 2 percent.

What we claim:

1. A piasrm the roaiiion ar artisansglucosidases, comprising cultivation of strains selected from the group consisting of Klebsielia rhinoscleromatis ATCC 13884, Klebsiella ozaenae ATCC 13885, :Klcbsiella edwardsii ATCC 13886 and Klebsiella rubiacearum ATCC 15574 on media consisting essentially of carbon sources, nitrogen sources, inorganic '7 8 salts under aerating and agitating conditions, accumu- 1,6-glucosidase is separated from the culture medium lation of alpha-1,6-glucosidases on the media, separ by salting out the culture broth against ammonium sultion of said glucosidases from the media and purificafate (02 Saturation) after conducing auto|yza of Said glucosidases' tion of the culture medium.

2. A process according to Claim 1 wherein the alphak w w w 

2. A process according to claim 1 wherein the alpha-1,6-glucosidase is separated from the culture medium by salting out the culture broth against ammonium sulfate (0.2 - 0.7 saturation) after conducting autolyzation of the culture medium. 